New non-viral gene transfer procedures are needed for human gene therapy in order to achieve long-term maintenance and expression of newly introduced genes. The purpose of this project is to continue evaluation and validation of the Sleeping Beauty (SB) transposon system for its efficacy to catalyze integration of transgenes to cure mucopolysaccharidosis diseases in murine and canine model systems. In the past few years we have demonstrated our ability to deliver both 2-glucuronidase (GUSB) and a-L-iduronidase (IDUA) encoding genes to mouse liver and thereby correct accumulation of storage vacuoles in non-neural tissues of MPS I mice. Here, we propose to extend these studies in order to determine the efficacy of the SB transposon system as a gene transfer vector in the mouse brain and in the liver of a larger animal as a prelude to scale-up for human gene therapy. Our results indicate that hydrodynamic injection of therapeutic transposons is the best method for non-viral gene delivery to livers of adult mice for long-term expression. We hypothesize that this procedure also can be efficacious in humans if applied to individual organs or tissues in a relatively non-invasive manner using catheter delivery. Accordingly, the project has three sub-goals. The first will employ the mouse model system: 1) Examine the duration of expression of IDUA following delivery of SB transposon vectors in the presence of immune responses (Aim1). The second is to develop an effective delivery of SB transposons into dog liver (Aims 2 and 3). The third sub-goal is to examine the subsequent duration of expression of GUSB and the effects in the MPS VII dog model (Aim 4). We will examine the physiological, neurological, and behavioral effects of expression of GUSB in enzyme-deficient animals, as well as inventory the integration site preferences of the transposon vector following hydrodynamic delivery into chromosomes of dog liver to help evaluate potential risks from insertional mutagenesis. The experiments in this project will provide an assessment of the capacity of SB as a gene-transfer vector to target therapeutically important organs in an in vivo gene therapy protocol. The results will lay the groundwork for its future application to human gene therapy of a variety of diseases, e.g., several MPS diseases and hemophilia, which can be treated by gene delivery to the liver. PUBLIC HEALTH RELEVANCE: Mucopolysaccharidoses (MPS) are medical conditions caused by deficiencies in any of several enzymes that result in physiological abnormalities and mental retardation. Recombinant enzyme replacement therapy is available but treatment costs can range from $100,000 to $1,000,000 per year. The goal of this grant is to determine whether transposons containing therapeutic genes can cure MPS disease in dogs as a model for human gene therapy.